ACTIVATION OF PROTEIN-KINASE-C BY PURIFIED BOVINE BRAIN 14-3-3 - COMPARISON WITH TYROSINE-HYDROXYLASE ACTIVATION

In the course of the purification of 14-3-3 protein (14-3-3) we found that 14-3-3 isolated from bovine forebrain activates protein kinase C (PKC), rather than the previously reported protein kinase C inhibitory activity (KCIP). We have characterized the 14-3-3 activation of PKC. The physical properties of purified PKC activator are the same as thos e previously reported for 14-3-3 and KCIP; i.e., (1) it is composed of subunits of molecular weight 32,000, 30,000, and 29,000; (2) it is ho mogeneous with respect to molecular weight, as judged by native gradie nt-gel electrophoresis, with a molecular weight of 53,000; and (3) it is composed of at least six isoforms when analyzed by reverse-phase HP LC. The concentration dependence of PKC activation by 14-3-3 is in the same range as that shown previously for KCIP inhibition of PKC, and a s that required for 14-3-3 activation of tyrosine hydroxylase; a maxim al stimulation of two- to three-fold occurs at 40-100 mu g/ml. 14-3-3' s activation of PKC is sensitive to alpha-chymotrypsin digestion but i s not heat labile. Activation is specific to PKC; at least two other p rotein kinases, cyclic AMP- and calcium/calmodulin-dependent protein k inases, are not activated. The activation of PKC by 14-3-3 is independ ent of phosphatidylserine and calcium and, as such, is an alternative mechanism for the activation of PKC that obviates its translocation to membranes.